Asthma has attracted a great deal of attention from both the public and from the medical community in the past few years. It has been termed an xe2x80x9cepidemic,xe2x80x9d and been the subject of cover stories in major newspapers and magazines. This is primarily due to the observation that the disease is worsening, particularly in Western, industrialized nations. In the past three decades, the prevalence, severity, and mortality of asthma have increased significantly. A recent study estimated the total annual cost in the United States at almost $6 billion. Once thought to be due to airway muscle spasm, asthma is now known to be an inflammatory disorder; during an asthma exacerbation, inflammation precedes bronchospasm. In acute asthma, eosinophils may form up to half of the cellular infiltrate, and bronchoalveolar eosinophilia invariably follows allergen inhalation in asthma attacks. Eosinophils cause inflammation and bronchial hyperreactivity through release of mediators such as leukotrienes, major basic protein, eosinophilic cationic protein, and eosinophilic peroxidase (Bruijnzeel, Ann NY Acad Sci 725, 259-267, (1994)).
Numbers and activity of eosinophils are controlled by cytokines released from activated T cells, especially IL-4, IL-5, and IL-13. T-lymphocytes can be divided on the basis of cytokine production, into Th1 and Th2 (Mosmann, T. R. et al., J. Immunol., 136, 2348-2357, (1986)). Th1 cells produce IL-2 and IFN-xcex3, but no IL-4 or IL-5, and Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 but no IL-2 or IFN-xcex3. Th1 and Th2 cells interact in a counterregulatory fashion: IL-4 and IL-10 promote Th2 development (Parronchi et al., J. Immunol., 149, 2977-2983 (1992), Swain et al., J. Immunol., 145, 3796-3806 (1990)) and inhibit Th1 cell and cytokine production (Moore, K. W. et al., Science, 248, 1230-1234 (1990)), and IFN-xcex3 inhibits the proliferation of Th2 cells (Gajewski et al., J. Immunol., 140, 4245-4252 (1988)) and promotes the development of Th1 cells (Parronchi et al., J. Immunol., 149 2977-2983 (1992)). IL-12, mainly a product of activated macrophages, is also a strong promoter of Th1 responses (Bliss, et al., J. Immunol., 156, 887-894 (1996)) and is often considered a Th1 cytokine; many of the activities ascribed to IL-12 are due to induction of IFN-xcex3. Th1 and Th2 cells have been identified in humans, in vivo as well as in vitro.
The Th2 cytokines, IL-4, IL-5, and IL-13 (Grunig, et al., Science, 282, 2261-2263 (1998), Robinson et al., N. Engl. J. Med., 326, 298-304 (1992), Wills-Karp et al., Science, 282, 2258-2261 (1998)), have been increasingly implicated in the inflammation of asthma. IL-4 amplifies allergic responses by inducing IgE production by uncommitted B-cells (Del Prete, et al., J. Immunol., 140, 4193-8 (1988)) and is a growth factor for mast cells (Saito, et al., Proc. Natl. Acad. Sci. USA, 85, 2288-2292 (1988)). IL-5 also stimulates immunoglobulin secretion (Swain, et al., J. Immunol., 145, 3796-3806 (1990), Takatsu et al., J. Immunol., 124, 2414-2422 (1980)) as well as stimulating the proliferation and activation of eosinophils (Clutterbuck, et al., Blood 73, 1504-1512 (1988), Lopez et al., J. Exp. Med., 167, 219-223 (1988), Walsh et al., Immunol., 71, 258-265 (1990)) and basophils (Hirai, K. et al., J. Exp. Med., 172, 1525-1528 (1990)). IL-13 has recently been shown to cause airway hyperresponsiveness and inflammation independently of eosinophils or IL-4 (Grunig et al., Science, 282, 2261-2263 (1998), Wills-Karp et al., Science, 282, 2258-2261 (1998)). In vitro, allergen-specific T-cell clones from atopic donors release Th2 cytokines after stimulation by specific allergens (Parronchi et al., Proc. Natl. Acad. Sci. USA, 88, 4538-4542 (1991)). During asthma exacerbations, peripheral T-cell activation and increased serum IL-5 correlate with eosinophilia and asthma symptoms (Corrigan et al., Am. Rev. Respir. Dis., 147, 540-547 (1993)), and bronchoalveolar lavage (BAL) T-cells release cytokines in a Th2-like pattern (Robinson et al., J. All. Clin. Immunol., 92, 313-324 (1993)), Robinson et al., N. Engl. J. Med., 326, 298-304 (1992), Robinson et al., J. All. Clin. Immunol., 92, 397-403 (1993)). Non-atopic asthmatics also have increased levels of Th2-like cytokines; increased IL-5 release from BAL T-lymphocytes is characteristic of both atopic and non-atopic asthmatics (Walker et al., Am. Rev. Respir. Dis., 146, 109-115 (1992)), and expression of peripheral T-cell IL-5 mRNA from non-atopic asthmatic subjects correlates with increased BAL (Marini et al., Chest 102, 661-669 (1992)) and peripheral blood (Walker et al., Am. Rev. Respir. Dis., 146, 109-115 (1992)) eosinophilia.
Because of these observations, the focus of treatment in asthma has shifted from primarily addressing bronchospasm, to one of modulating inflammation. Recent guidelines to the management of asthma recommend that anti-inflammatory therapy be used for all but the most intermittent and benign cases of the disease. Current anti-inflammatory therapy, however, remains disappointingly broad and nonspecific; corticosteroids are the xe2x80x9cgold standardxe2x80x9d for asthma treatment, and inhaled corticosteroids are only incrementally better than they were 25 years ago. The much-touted leukotriene pathway antagonists which have been released in the last five years have been helpful only in a subset of asthmatics.
Positive tubercullin skin tests are associated with protection against atopy and asthma (Shirakawa et al., Science, 275, 77xe2x89xa079 (1997)) as well as systemic Th1 responses; however, the antigen(s) responsible for this protection has not been identified. The Fibronectin Attachment Protein of Mycobacterium bovis-BCG (FAP-B) has been identified and cloned. FAP-B is responsible for binding of the organism to fibronectin and for epithelial entry. Other FAPs have been isolated (M. vaccae FAP-V) and in some cases cloned (M. leprae FAP-L and M. avium FAP-A). Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin.
Surprisingly and unexpectedly, the FAP-B polypeptide is capable of protecting against the induction of an atopic/asthmatic inflammatory response. FAP-B and related polypeptides offer potential therapeutic benefit in asthma. Without intending to be bound by theory, it is expected that the mechanism of action through which FAP-B offers protection against Th2-mediated responses is through induction of Th1 responses, such as IFN-xcex3 and IL-12, which can downregulate Th2 responses.
Accordingly, the present invention provides methods for treating at least one symptom of an inflammatory response in a mammal. In one embodiment, a method includes administering an effective amount of a microbial polypeptide to the mammal such that at least one symptom of an inflammatory response, for instance a Th2 mediated inflammatory response, is inhibited. Optionally, the microbial polypeptide is isolated. The microbial polypeptide can be administered prior to exposure to at least one suspected or known inflammation response-inducing agent, or administered during or after exposure to at least one suspected or known inflammation response-inducing agent.
The microbial polypeptide can be encoded by a nucleotide sequence, where the complement of the nucleotide sequence hybridizes to the nucleotide sequence set forth at nucleotides 79 to 1056 of SEQ ID NO:1 in a solution containing 50% formamide, 6xc3x97SSC, 7xc3x97Denhardt""s reagent, 0.7% SDS, 150 xcexcg/ml salmon sperm DNA at 42xc2x0 C. for at least about 12 hours, followed by one wash for 30 minutes at 25xc2x0 C. in a solution containing 1xc3x97SSC, one wash for 30 minutes at 42xc2x0 C. in a solution containing 1xc3x97SSC, and one wash for 30 minutes at 42xc2x0 C. in a solution containing 0.1xc3x97SSC. For instance, the nucleotide sequence encoding the microbial polypeptide can be nucleotides 79 to 1056 of SEQ ID NO:1. The microbial polypeptide can also inhibit fibronectin attachment to bacillus Calmette-Guerin in a dose-dependent manner.
The microbial polypeptide can be a Mycobacterial polypeptide. The amino acid sequence of the Mycobacterial polypeptide can be the amino acid sequence of SEQ ID NO:2, GenBank accession AAB34676, GenBank accession AAB50543, GenBank accession CAA56555, GenBank accession AAB36458, GenBank accession P46842, or active analogs and active fragments thereof. Alternatively, the amino acid sequence of the Mycobacterial polypeptide can include amino acids 47 to 325 of SEQ ID NO:2, amino acids 112 to 283 of SEQ ID NO:2, 121 to 283 of SEQ ID NO:2, or active analogs and active fragments thereof. In another alternative, the amino acid sequence of the Mycobacterial polypeptide can be SEQ ID NO:3 and amino acids 47 to 325 of SEQ ID NO:2, and active analogs and active fragments thereof, wherein the carboxy terminal amino acid of SEQ ID NO:3 is fused to the amino terminal amino acid of amino acids 47 to 325 of SEQ ID NO:2.
The inflammatory response can be associated with a disease including skin allergy, hives, allergic rhinitis, conjunctivitis, hay fever, asthma, or allergic gastroenteritis. The types of asthma include extrinsic asthma and intrinsic asthma. When the asthma is extrinsic, it can be allergic asthma, occupational asthma, and allergic bronchopulmonary aspergillosis. When the asthma is intrinsic, it can be associated with an irritant including a pathogen, for instance a pathogen that causes a respiratory tract infection in the mammal, or an inhaled pollutant.
Another aspect of the invention provides a method for treating at least one symptom of an inflammatory response, including asthma, in a mammal including administering to the mammal a polypeptide such that at least one symptom of the inflammatory response is inhibited. The amino acid sequence of the polypeptide is SEQ ID NO:3 and amino acids 47 to 325 of SEQ ID NO:2, and active analogs and active fragments thereof, where the carboxy terminal amino acid of SEQ ID NO:3 is fused to the amino terminal amino acid of amino acids 47 to 325 of SEQ ID NO:2.
The present invention also provides an inflammation reaction inhibiting composition that includes a microbial polypeptide, for instance a Mycobacterial polypeptide, and a pharmaceutically acceptable carrier. In one embodiment, the polypeptide can comprise an amino acid sequence of SEQ ID NO:3 and amino acids 47 to 325 of SEQ ID NO:2, and active analogs and active fragments thereof, where the carboxy terminal amino acid of SEQ ID NO:3 is fused to the amino terminal amino acid of amino acids 47 to 325 of SEQ ID NO:2. In an alternative embodiment, the polypeptide is encoded by a nucleotide sequence, where the complement of the nucleotide sequence hybridizes to the nucleotide sequence set forth at nucleotides 79 to 1056 of SEQ ID NO:1 in a solution containing 50% formamide, 6xc3x97SSC, 7xc3x97Denhardt""s reagent, 0.7% SDS, 150 xcexcg/ml salmon sperm DNA at 42xc2x0 C. for at least about 12 hours, followed by one wash for 30 minutes at 25xc2x0 C. in a solution containing 1xc3x97SSC, one wash for 30 minutes at 42xc2x0 C. in a solution containing 1xc3x97SSC, and one wash for 30 minutes at 42xc2x0 C. in a solution containing 0.1xc3x97SSC.
The present invention further provides a method for treating symptoms of an inflammatory response in a mammal, including administering an effective amount of a polypeptide to the mammal such that at least one symptom of an inflammatory response is inhibited. The polypeptide is encoded by a nucleotide sequence, where the complement of the nucleotide sequence hybridizes to the nucleotide sequence set forth at nucleotides 79 to 1056 of SEQ ID NO:1 in a solution containing 50% formamide, 6xc3x97SSC, 7xc3x97Denhardt""s reagent, 0.7% SDS, 150 xcexcg/ml salmon sperm DNA at 42xc2x0 C. for at least about 12 hours, followed by one wash for 30 minutes at 25xc2x0 C. in a solution containing 1xc3x97SSC, one wash for 30 minutes at 42xc2x0 C. in a solution containing 1xc3x97SSC, and one wash for 30 minutes at 42xc2x0 C. in a solution containing 0.1xc3x97SSC.
Definitions
xe2x80x9cTreatingxe2x80x9d refers to the administration of a polypeptide or a composition that includes a polypeptide at any time prior to the onset of at least one symptom of an inflammatory response, i.e., prophylactic therapy. xe2x80x9cTreatingxe2x80x9d also refers to the administration of a polypeptide or a composition that includes a polypeptide during or after the onset of at least one symptom of an inflammatory response to ameliorate at least one symptom of an inflammatory response. In other words, xe2x80x9ctreatingxe2x80x9d refers to both the prevention (prophylactic) and to the amelioration (therapeutic) of at least one symptom of an inflammatory response.
Inflammatory response, and symptoms of an inflammatory response are described in greater detail herein. An inflammatory response-inducing agent is a substance that induces an inflammatory response in a mammal. Non-limiting examples of inflammatory response-inducing agents include allergens, particulates, pathogens, and pollutants such as tobacco smoke. xe2x80x9cExposurexe2x80x9d to inflammatory response-inducing agents indicates that a mammal is in an environment where inflammatory response-inducing agents are present or may be present.
An xe2x80x9ceffective amountxe2x80x9d of a polypeptide refers to an amount of the polypeptide that is sufficient to inhibit in a mammal at least one symptom of an inflammatory response.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. A polypeptide can be produced by an organism, or produced using recombinant techniques, or chemically or enzymatically synthesized. A xe2x80x9cmicrobial polypeptidexe2x80x9d refers to a polypeptide that is expressed by a microbe, is encoded by a coding region isolated from a microbe, is encoded by a coding region that hybridizes with a nucleotide sequence as described in greater detail herein or that has a certain percentage structural similarity with a nucleotide sequence as described in greater detail herein, or has a certain percentage structural similarity with a polypeptide as described herein. A type of microbial polypeptide is xe2x80x9cmycobacterial polypeptide,xe2x80x9d where xe2x80x9cmycobacterialxe2x80x9d refers to a strain of the genus Mycobacterium. A coding region refers to a polynucleotide that encodes a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding region are generally determined by a translation start codon at its 5xe2x80x2 end and a translation stop codon at its 3xe2x80x2 end.
An active analog or active fragment of a polypeptide is one that retains the ability to treat the symptoms of an inflammatory response in an animal as described herein. Active analogs and active fragments are described in greater detail herein.
The term xe2x80x9ccomplementxe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d as used herein, refers to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one polynucleotide will base pair to a thymine on a second polynucleotide and a cytosine on one polynucleotide will base pair to a guanine on a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5xe2x80x2-ATGC and 5xe2x80x2-GCAT are complementary. The terms complement and complementary also encompass two polynucleotides where one polynucleotide contains at least one nucleotide that will not base pair to at least one nucleotide present on a second polynucleotide. For instance the third nucleotide of each of the two polynucleotides 5xe2x80x2-ATTGC and 5xe2x80x2-GCTAT will not base pair, but these two polynucleotides are complementary as defined herein. Typically two polynucleotides are complementary if they hybridize under certain conditions.
As used herein, xe2x80x9chybridizes,xe2x80x9d xe2x80x9chybridizing,xe2x80x9d and xe2x80x9chybridizationxe2x80x9d means that a single stranded polynucleotide forms a noncovalent interaction with a complementary polynucleotide under certain conditions, as described herein.
Unless noted otherwise, a xe2x80x9cpathogenxe2x80x9d as used herein refers to a virus or a microbe, including prokaryotic microbes and eukaryotic microbes, that is capable of causing a respiratory tract infection in a mammal, whether the mammal is immunocompromised or not. The terms xe2x80x9cbacillus Calmette-Guerinxe2x80x9d and M. bovis BCG are used interchangeably and refer to a strain of M. bovis that has been rendered completely avirulent.
An xe2x80x9cisolatedxe2x80x9d polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities.